The present invention is generally in the field of detecting analytes in a medium and, more particularly, the present invention relates to the methods of making optical diffraction-based sensors which are capable of indicating the presence of the analyte in a medium.
There are many systems and devices available for detecting a wide variety of analytes in various media. Most of these systems and devices are relatively expensive and require a trained technician to perform the test. There are many cases where it would be advantageous to be able to rapidly and inexpensively determine if an analyte were present. What is needed is a biosensor system that is easy and inexpensive to manufacture and is capable of reliable and sensitive detection of analytes, including smaller analytes.
Sandstrom et al., 24 Applied Optics 472, 1985, describe use of an optical substrate of silicon with a layer of silicon monoxide and a layer of silicon formed as dielectric films. They indicate that a change in film thickness changes the properties of the optical substrate to produce different colors related to the thickness of the film. The thickness of the film is related to the color observed and a film provided on top of an optical substrate may produce a visible color change. The authors indicate that a mathematical model can be used to quantitate the color change, and that xe2x80x9c[c]alculations performed using the computer model show that very little can be gained in optical performance from using a multilayer structure . . . but a biolayer on the surface changes the reflection of such structures very little since the optical properties are determined mainly by the interfaces inside the multilayer structure. The most sensitive system for detection of biolayers is a single layer coating, while in most other applications performance can be by additional dielectric layers.xe2x80x9d
Sandstrom et al., go on to indicate that slides formed from metal oxides on metal have certain drawbacks, and that the presence of metal ions can also be harmful in many biochemical applications. They indicate that the ideal top dielectric film is a 2-3 nm thickness of silicon dioxide which is formed spontaneously when silicon monoxide layer is deposited in ambient atmosphere, and that a 70-95 nm layer silicon dioxide on a 40-60 nm layer of silicon monoxide can be used on a glass or plastic substrate. They also describe formation of a wedge of silicon monoxide by selective etching of the silicon monoxide, treatment of the silicon dioxide surface with dichlorodimethylsilane, and application of a biolayer of antigen and antibody. From this wedge construction they were able to determine film thickness with an ellipsometer, and note that the xe2x80x9cmaximum contrast was found in the region about 65 nm where the interference color changed from purple to blue.xe2x80x9d They indicate that the sensitivity of such a system is high enough for the detection of protein antigen by immobilized antibodies. They conclude xe2x80x9cthe designs given are sensitive enough for a wide range of applications. The materials, i.e., glass, silicon, and silicon oxides, are chemically inert and do not affect the biochemical reaction studied. Using the computations above it is possible to design slides that are optimized for different applications. The slides can be manufactured and their quality ensured by industrial methods, and two designs are now commercially available.
U.S. Pat. No. 5,512,131 issued to Kumar et al. describes a device that includes a polymer substrate having a metal coating. An analyte-specific receptor layer is stamped on the coated substrate. The device is used in a process for stamping or as a switch. A diffraction pattern is generated when an analyte binds to the device. A visualization device, such as a spectrometer, is then used to determine the presence of the diffraction pattern.
However, the device described by Kumar et al. has several disadvantages. One disadvantage is that an extra visualization device is needed to view any diffraction pattern. By requiring a visualization device, the Kumar et al. device does not allow a large number of samples to be tested since it is not possible to determine the presence of an analyte by using the unaided eye. Additionally, this device is not able to detect smaller analytes as these analytes do not produce a noticeable diffraction pattern.
U.S. Pat. No. 5,482,830 to Bogart, et al., describes a device that includes a substrate which has an optically active surface exhibiting a first color in response to light impinging thereon. This first color is defined as a spectral distribution of the emanating light. The substrate also exhibits a second color which is different from the first color (by having a combination of wavelengths of light which differ from that combination present in the first color, or having a different spectral distribution, or by having an intensity of one or more of those wavelengths different from those present in the first color). The second color is exhibited in response to the same light when the analyte is present on the surface. The change from one color to another can be measured either by use of an instrument, or by eye. Such sensitive detection is an advance over the devices described by Sandstrom and Nygren, supra, and allow use of the devices in commercially viable and competitive manner.
However, the method and device described in the Bogart, et al. patent has several disadvantages. One disadvantage is the high cost of the device. Another problem with the device is the difficulty in controlling the various layers that are placed on the wafer so that one obtains a reliable reading.
Additionally, biosensors having a self-assembling monolayer have been used to detect analytes and are set forth in U.S. patent application Ser. Nos. 08/768,449 and 08/991,844, both of which are incorporated herein by reference in their entirety. However, these biosensors currently do not have the requisite sensitivity required to detect smaller analytes since these smaller analytes do not produce a sufficient diffraction pattern to be visible.
Some commercial lateral flow technologies have been used which employ latex bead technology. These technologies are currently employed in most of the commercially-available home diagnostic kits (e.g. pregnancy and ovulation kits). These kits use colored beads which accumulate in a defined xe2x80x9ccapture zonexe2x80x9d until the amount of beads becomes visible to the unaided eye. However, these systems lack the requisite sensitivity to test for many analytes, since a much larger number of latex beads must bind in the capture zone to be visible to the naked eye than that required to cause diffraction in the same size zone. Theoretically, the number of beads needed is about 2 to 3 orders of magnitude higher than the sensors of the present invention.
Biosensors having a self-assembling monolayer and using microparticle technology have been used to detect smaller analytes and are set forth in U.S. patent application Ser. No. 09/210,016, which is incorporated herein by reference in its entirety. However, these biosensors require multiple process steps to produce, thereby increasing the difficulty and cost for using these types of sensors.
What is needed is a biosensor system that is easy and inexpensive to manufacture and is capable of reliable and sensitive detection of analytes, including smaller analytes.
The present invention provides an inexpensive and sensitive system and method for detecting analytes present in a medium. The invention provides a new approach to reduce the number of steps involved by a user of diffraction diagnostic devices using xe2x80x9cdiffraction enhancing elements,xe2x80x9d such as microparticles. The approach involves the use of a wicking agent that is used to remove unbound labeled microparticles, as well as any residual liquid from the sample. The wicking agent then avoids the necessity of any additional rinsing, which may be cumbersome or more difficult for a user. Additionally, a small hole (e.g., {fraction (3/32)} of an inch) can be punched out of the center of the wicking agent so that once the sample and excess particles are wicked away, the hole allows the user to immediately check for a diffraction image without removing the wicking material. Examples of wicking agents include nitrocellulose membranes, cellulose acetate membranes, and glass microfiber structures.
In addition, the pore size of the membrane may be varied to control the rate and force of wicking. This can affect the accuracy of the diagnostic device, and can also be taken advantage of to create a one-step device. To achieve this, the one-step device consists of the contact printed capture antibody on a substrate, such as the gold/MYLAR(copyright), which then has labeled particles pre-dried onto its surface. Additionally, a small pore size membrane (e.g., 0.45 micron nitrocellulose) with a hole cut out is placed on top of the device to complete it. The user simply adds the sample (e.g., serum or blood) to be tested, and then views for a diffraction-image once wicking has occurred. The small pore size delays wicking long enough to allow adequate incubation, such as that needed for antibody-antigen interactions to take place. Alternatively, wicking may be delayed by using an erodible reagent at the periphery of the wicking agent circle. The reagent would eventually dissolve or derivatize so that it would allow wicking after a specific time period
The system of the present invention is much more sensitive than current inexpensive systems. The system of the present invention is able to detect low to high molecular weight analytes, microorganisms, and DNA or RNA species in fluid samples. More specifically, the system is able to detect hormones, steroids, antibodies, drug metabolites, and even nucleic acids, among others. This is a significant expansion of the optical diffraction-based sensing technology set forth in U.S. patent application Ser. Nos. 08/768,449 and 08/991,844.
The present invention utilizes diffraction enhancing elements, such as latex microspheres, which aid in the detection of smaller analytes. Normally, after an analyte binds to an analyte-specific receptor on a biosensor, the analyte will diffract or reflect transmitted light to produce a diffraction pattern. If the analyte is larger, the diffraction pattern is able to be seen with the unaided eye. However, some analytes are too small such that the diffraction pattern produced is not able to be seen. By using diffraction enhancing elements, the biosensor having the analyte-specific receptor material may be used to detect these smaller analytes. The diffraction enhancing elements used are capable of binding to the analyte, and then the element with bound analyte binds to the biosensor. Then, as the light is transmitted through or reflected from the biosensor, the element enhances the diffraction pattern generated by the analyte such that the resulting diffraction pattern may be seen by the unaided eye.
The present invention also utilizes methods of contact printing of patterned, analyte-specific receptors. The analyte-specific receptors have receptive materials bound thereto. The receptive materials are specific for a particular analyte or class of analyte, depending upon the receptor used. Methods of contact printing which would be useful in generating the sensing devices used in the present system are disclosed fully in U.S. patent application Ser. Nos. 08/707,456 and 08/769,594, both of which are incorporated herein by reference in their entirety. However, since these methods relate to self-assembling monolayers, the methods need to be altered slightly, as discussed below, to print the analyte-specific receptor material as this material is not self-assembling.
Patterned analyte-specific receptor layers allow for the controlled placement of analytes with or without diffraction enhancing elements thereon via the patterns of analyte-specific receptors. The biosensing devices of the present invention produced thereby are used by first exposing the biosensing device to the sample medium (that may or may not contain the analyte of choice) mixed with the diffraction enhancing element. Then, after an appropriate incubation period, a light, such as a laser or other point light source, is transmitted through or reflected from the film. If the analyte is present in the medium and is bound, either directly or in conjunction with the diffraction enhancing element, to the receptors on the patterned analyte-specific receptor layer, the light is diffracted in such a way as to produce a visible image. In other words, the analyte-specific receptor layers with the analyte and/or diffraction enhancing element bound thereto can produce optical diffraction patterns which differ depending on the reaction of the receptors on the analyte-specific receptor layer with the analyte of interest. The light can be in the visible spectrum, and be either reflected from the film, or transmitted through it, and the analyte can be any compound or particle reacting with the analyte-specific receptor layer. The light can be a point white light source or monochromatic electromagnetic radiation in the visible region. While visible light is the desired light source, the present invention may also be used with non-visible point light sources, such as near-infrared light, coupled with a detector. The thickness of the film and the size of the microparticle may be adjusted to compensate for the non-visible light source. Additionally, the present invention also provides a flexible support for an analyte-specific receptor layer either directly on the substrate or on gold or other suitable metal or metal alloy.
The present invention provides an analyte-specific receptor layer on gold or other material which is suitable for mass production. The biosensors used in the present invention can be produced as a single test for detecting an analyte or it can be formatted as a multiple test device. The biosensors of the present invention can be used to detect (1) antigens or antibodies associated with medical conditions, (2) contamination in garments, such as diapers, and (3) contamination by microorganisms.
In another embodiment of the present invention, nutrients for a specific class of microorganisms can be incorporated into the analyte-specific receptor layer. In this way, very low concentrations of microorganisms can be detected by first contacting the biosensor of the present invention with the nutrients incorporated therein and then incubating, if necessary, the biosensor under conditions appropriate for the growth of the bound microorganism. The microorganism is allowed to grow until there are enough organisms to form a diffraction pattern.
These and other features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments.